NF-κB-HOTAIR axis links DNA damage response,

ORIGINAL ARTICLENF-κB-HOTAIR axis links DNA damage response,chemoresistance and cellular senescence in ovarian cancerAR Öze ş1,DF Miller 2,ON Öze ş3,F Fang 2,Y Liu 4,5,6,D Matei 6,7,8,T Huang 9and KP Nephew 1,2,6,8,10INTRODUCTIONThe mammalian DNA damage response (DDR)to genotoxic stress is critically important for maintaining genome stability,cell survival and preventing cellular transformation.DDR networks include the classic tumor suppressor gene p53and its down-stream target p21.1More recently,a DDR mechanism has been reported involving nuclear factor kappa B (NF-κB),a master regulator of over 400genes involved in in flammation,apoptosis,cell-cycle control and cell senescence.2,3NF-κB signaling-mediated activation of DDR has been shown to restore genomic integrity,augment cancer cell survival and have a role in the development of resistance to platinum-based cancer therapies.4,5However,distinct DNA damage-induced NF-κB signaling pathways contributing to chemoresistance in cancer have not been well studied.Platinum,the first line of therapy for ovarian and other cancers,induces inter-and-intra strand crosslinks,generates single-stranded breaks and activates nucleotide excision repair.6Cellular responses to DNA damage,including response to platinum,can be regulated at the post-transcriptional level by long non-coding RNAs (lncRNAs).7The JADE1adjacent regulatory RNA (lncRNA-JADE)was reported to have a crucial role in DNA damage-induced,histone H4acetylation associated with transcriptional activation,8and lncRNA-p21was shown to physically interact with hnRNPK (heterogenous nuclear ribonuclear proteins)during DDR and mediate p53-dependent transcriptional repression.9Platinum-induced DNA damage resulted in lncRNA PANDA (p21-associated ncRNA DNA damage activated)activation,interaction withtranscription factor NF-YA and PANDA-NF-YA modulation of p53-dependent apoptosis.10HOX antisense intergenic RNA (HOTAIR),an lncRNA frequently overexpressed in human cancers,11was originally identi fied in 2007by Rinn et al .as an lncRNA located in the HOXC cluster on chromosome 12that regulates the HOXD gene cluster on chromosome 2.12HOTAIR transcriptionally silences genes located on a distant chromosome region through an epigenetic mechan-ism involving interaction with polycomb repressive complex 2(PRC2).7This interaction appears to be required for PRC2occupancy of speci fic loci,such as the HOXD locus,H3K27me3(trimethylation of histone H3lysine K27)by enhancer of zeste 2(EZH2)and subsequent gene repression.12,13Additional inter-chromosomal targets of HOTAIR include cancer-associated genes such as protocadherin (PCDH),ephrin receptor (EPHA1)and the NF-κB inhibitory protein I κ-B α.12,14Although epigenetic processes have been reported to have critical roles in DDR,15including aberrant histone methylation by EZH2,16,17a direct role for HOTAIR in DDR has not been investigated.However,functional overlap between PRC2and NF-κB activation by genotoxic agents,in flammation and cancer has been reported,18suggesting a potential interaction between NF-κB activation and HOTAIR in response to DNA damage.Furthermore,in solid tumors,including ovarian cancer (OC),enhanced NF-κB activation has been observed in aggressive chemoresistant cell lines.19The objective of this study was to investigate the functional role for HOTAIR in DDR and chemotherapy resistance.We demonstrate a strong correlation of HOTAIR overexpression with platinum1Molecular and Cellular Biochemistry Department,Indiana University,Bloomington,IN,USA;2Medical Sciences Program,Indiana University School of Medicine,Bloomington,IN,USA;3Department of Medical Biology and Genetics,Akdeniz University,Antalya,Turkey;4Department of Medical and Molecular Genetics,Indiana University School of Medicine,Indianapolis,IN,USA;5Center for Computational Biology and Bioinformatics,Indianapolis,IN,USA;6Indiana University Melvin and Bren Simon Cancer Center,Indianapolis,IN,USA;7Department of Medicine,Indiana University School of Medicine,Indianapolis,IN,USA;8Department of Obstetrics and Gynecology,Indiana University School of Medicine,Indianapolis,IN,USA;9Department of Molecular Medicine/Institute of Biotechnology,The University of Texas Health Science Center at San Antonio,San Antonio,TX,USA and 10Department of Cellular and Integrative Physiology,Indiana University School of Medicine,Indianapolis,IN,USA.Correspondence:Dr KP Nephew,Department of Cellular and Integrative Physiology,Medical Sciences Program,Indiana University School of Medicine,Jordan Hall 302,1001East Third Street,Bloomington,47405-4401IN,USA.E-mail:knephew@Received 20August 2015;revised 16December 2016;accepted 4January 2016Oncogene (2016),1–12©2016Macmillan Publishers Limited All rights reserved 0950-9232//oncresistance in OC cell lines and patient tumors.In response to DNA damage,we found that NF-κB directly upregulates HOTAIR expression in OC cell lines.Downregulation of Iκ-Bαduring DDR induced an NF-κB-HOTAIR signaling positive-feedback loop cascade,and we demonstrate that DDR further induces HOTAIR-mediated expression of p65-NF-κB and NF-κB target genes matrix metalloprotease-9(MMP-9)and interleukin-6(IL-6)to promote OC cellular senescence and resistance to DNA-damaging agents. Collectively,these results are thefirst to demonstrate a role for HOTAIR in DNA damage-induced NF-κB signaling pathway, identifying HOTAIR as a new therapeutic target in drug-resistant OC and likely other cancers.RESULTSHOTAIR is overexpressed in drug-resistant OCWe examined expression of HOTAIR in a panel of OC cell lines (Figure1a),representing a spectrum of platinum(CDDP)sensitivity (Resistant;Sensitive).Increased(P o0.05)HOTAIR expression was observed in CDDP-resistant(IC50levels410μM)compared with -sensitive cell lines(Figure1a and Supplementary Table S1)and in OC tumors obtained from unpaired patients with CDDP-resistant vs-sensitive high-grade serous disease(2.1log2fold-change; Figure1b).We then examined patient data obtained from The Cancer Genome Atlas(TCGA;publically available expression and clinical annotation data).20HOTAIR expression wasincreasedFigure1.HOTAIR expression and characterization in OC cells.(a)HOTAIR expression in OC cell lines(IGROV,OVSAHO,OVMUNA,SKOV3, A2780,HEYC2,A2780-CR5,and OV90)was determined by qRT-PCR(values normalized to GAPDH).Values represent the average of three biological replicates.(b)HOTAIR expression in primary high-grade serous ovarian tumors from unpaired patient samples at initial diagnosis and platinum-sensitive(n=11)compared with patients with recurrent and platinum-resistant disease(n=14)(c)TCGA data analysis for HOTAIR Read Per Kilobase Million(RPKM)values in OC patients with primary vs recurrent disease.(d)Clonogenic growth in A2780p overexpressing HOTAIR(left and middle graphs)and A2780_CR5cells with dsiRNA-mediated depletion of HOTAIR.Graphs represent the fraction of surviving cells normalized to untreated population.Graphs represent triplicate experiments.(e)Xenograft tumor growth in athymic mice of A2780p expressing HOTAIR and treated with cisplatin(10μM CDDP)or vehicle control(2×106cells per injection).Area under the curve (AUC)was calculated at10weeks(n=4mice per group).Asterisks indicate P o0.05(*)or P o0.01(**).NF-κB-HOTAIR axis links DDR,chemoresistance and cellular senescenceARÖzeşet al2Oncogene(2016)1–12©2016Macmillan Publishers Limited(40.5log10fold-change)in OC patients with recurrent comparedwith primary high-grade serous disease(Figure1c). Overexpression of HOTAIR increases colony formation and CDDP resistanceTo further examine the association between HOTAIR expressionand chemoresistance,we either overexpressed(80-fold increasevs control;Supplementary Figure S1A)HOTAIR in CDDP-sensitiveA2780p or HEYC2(moderate HOTAIR levels)or knocked-down(70%reduction compared with dsiGFP control,SupplementaryFigure S1B)HOTAIR in CDDP-resistant A2780_CR5and examinedfunctional changes using clonogenic and proliferation assaysin vitro and a mouse xenograft model in vivo.HOTAIR over-expression increased(P o0.05)whereas HOTAIR knockdowndecreased(P o0.05)OC clonogenic survival(Figure1d andSupplementary Figure S1C).However,overexpression or knock-down of HOTAIR had no effect on cell doubling time(Supplementary Figure S1D and E),indicating that cell prolifera-tion rate was not altered.To test whether HOTAIR overexpressioncontributed to CDDP resistance in vivo,A2780p was transfectedwith either HOTAIR or vector control,treated with CDDP or vehicleto induce DNA damage,and then injected subcutaneously in nudemice(see Materials and methods).Tumor size was measuredbiweekly.Tumor growth,measured by assessing area under thecurve of the xenograft tumor,was greater(P o0.05)for HOTAIRoverexpressing OC tumors compared with control(Figure1e andSupplementary Figure S1F),indicating a role for HOTAIR over-expression in response to DNA-damaging agents and CDDPresistance.HOTAIR mediates DDR in OC cellsWe observed that CDDP treatment of A2780p cells increased(P o0.05)HOTAIR expression starting at16h post treatment(Supplementary Figure S2A)and that HOTAIR expression was alsoincreased by other DNA damage agents mitomycin C andhydrogen peroxide(MMC and H2O2;Supplementary Figure S2Band C).To further examine a role for HOTAIR as a potentialmediator of DDR,we examined known cellular responses togenotoxic stress and apoptosis21after platinum treatment,including phosphorylation of Chk1(p-Chk1),formation of γ-H2AX foci and caspase cleavage.A2780p cells overexpressing HOTAIR or A2780_CR5cells transfected with dsiRNA HOTAIR weretreated with CDDP.HOTAIR overexpression increased s317phosphorylation of Chk1,a marker for Chk1activation,inA2780p(Figure2a and Supplementary Figure S2D),whereasHOTAIR knockdown decreased Chk1s317phosphorylation inA2780_CR5(Figure2b and Supplementary Figure S2E).Interest-ingly,in the Kuramochi OC cell line harboring p53D281Y mutant(DNA binding mutant),phosphorylation of both p53and Chk1wasinduced by HOTAIR overexpression(Supplementary Figure S2F).No expression of p21was observed(data not shown),confirminginactive p53(Supplementary Table S1).p-Chk1levels correlatedwithγ-H2AX phosphorylation,and the percentage of cellscontaining45γ-H2AX foci increased(1.5-and4-fold)by1and24h of CDDP exposure in HOTAIR overexpressing A2780p cells(Figure2c)and decreased in A2780_CR5cells(2.3-and1.5-fold)by1and24h(Figure2d).The number ofγ-H2AX foci was unchangedin untreated cells(Supplementary Figure S2G).Furthermore,enforced overexpression of HOTAIR in A2780p decreased caspase3/7cleavage(Figure2e;left)and increased caspase3/7cleavagein A2780_CR5cells transfected with dsiRNA HOTAIR(Figure2e;right),an established characteristic of cells undergoing apoptosis,indicating that HOTAIR overexpression inhibited platinum-induced apoptosis.Overexpression of HOTAIR leads to specific changes in gene expressionTo investigate the association between HOTAIR,DDR and chemoresistance and identify genes and pathways affected by HOTAIR overexpression,we performed whole transcriptome RNA sequencing of A2780p(CDDP-sensitive)and A2780_CR5 (CDDP-resistant and endogenous HOTAIR overexpression)OC cell lines.Marked differences in overall gene expression profiles were observed between the paired lines(Supplementary Figure S3Aand B),and expression of DDR-associated genes(for example, MLH1and XRCC3),NF-κB pathway(for example,IL-6R,IL-4R,NCAMand BCL2L211)and epigenetic regulators(that is,PRC-associated genes)were prominently altered in A2780_CR5compared withA2780p(Supplementary Figure S3C).As expected,differential expression of cancer relevant cellular processes and pathways associated with chemoresistance(anti-apoptotic,cell adhesion,inflammation)in these cell lines was also observed,including theNF-κB pathway(Supplementary Figure S3B).Further examinationof the highly enriched NF-κB pathway by western blot analysisconfirmed that total p65nuclear levels were increased(3.2-fold vsA2780p)in A2780_CR5(Figure3a).To validate NF-κB activation by HOTAIR,we measured Iκ-Bαlevels in A2780p cells overexpressing HOTAIR treated with CDDP (Figure3b).We observed Iκ-Bαprotein levels were decreased indicating that by repressing the inhibitory Iκ-Bαunit,HOTAIR can activate NF-κB during DDR.Next,we measured nuclear p65-NF-κB expression in A2780p HOTAIR overexpressing and A2780_CR5 dsiHOTAIR knockdown cells using immunofluorescence and western blotting.We observed a twofold increase in NF-κB translocation1and24h post CDDP treatment in A2780cells (Figure3c),and an increase in nuclear p65and a decrease in both nuclear and cytoplasmic Iκ-Bαin the western blot(Figure3d). Additionally,we observed a threefold andfivefold decrease inNF-κB translocation1and24h post CDDP treatment inA2780_CR5cells(Figure3e)and a decrease in nuclear p65andan increase in cytoplasmic Iκ-Bαlevels with western blotting (Figure3f),indicating that HOTAIR expression promotes sustainedNF-κB activation during DDR.To further investigate the role of NF-κB transcriptional activity in stress(genotoxic)-induced HOTAIR expression(Supplementary Figure2A–C),A2780p cells were treated with CDDP.As shown in Figure4a,an increase in HOTAIR expression was observed (ninefold)at24h post CDDP treatment and co-treatment withan NF-κB inhibitor(Bay-11)inhibited(P o0.001)CDDP-induced HOTAIR expression.TNF-αtreatment increased(P o0.05)expres-sion of HOTAIR(16-fold by2h;Supplementary Figure4A)and ectopic overexpression of two known inducers of NF-κB,IKK-αandIKK-β,22increased(P o0.05)HOTAIR expression(3-fold;Figure4b). Furthermore,in A2780p ectopically overexpressing HOTAIR,NF-κB targets IL-6and MMP-9were upregulated(P o0.05),while expression of these genes was reduced(P o0.05)in HOTAIR-knockdown A2780_CR5(Figure4c).Moreover,in xenograft OC tumors overexpressing HOTAIR,CDDP treatment increased overall expression of HOTAIR and NF-κB target genes,with IL-6and MMP-9upregulation(P o0.05)relative to tumors derived from untreated OC cells(Figure4d),confirming functionality of the pathway in vivo and stable gene expression changes.Ourfindings that HOTAIR expression was increased hours post-DNA damage and this induced expression was blocked by inhibition of NF-κB activation(Supplementary Figure2A)indicated transcriptional regulation of the lncRNA in an NF-κB/DNA damage-dependent manner.To begin to determine the underlying mechanism,we used promoter-binding assays to examine the HOTAIR promoter region(1kb upstream of transcription start site (TSS)).We identified a putative p65-NF-κB binding site (906-GGGACACCCC-915)(Figure5a)and investigated NF-κB-p65 binding to the HOTAIR promoter region in A2780_CR5cells usingNF-κB-HOTAIR axis links DDR,chemoresistance and cellular senescenceARÖzeşet al3©2016Macmillan Publishers Limited Oncogene(2016)1–12chromatin immunoprecipitation (ChIP)with five different primer sets (1,10kb;2,8kb;3,4kb;4:900bp;5,200bp from the TSS)spanning 10kb upstream of the TSS (Figure 5b).Enrichment (fourfold)of p65in the canonical binding site (primers 4and 5)was observed compared with control primer 1(Figure 5b),further supporting a regulatory role for NF-κB pathway in HOTAIR expression.Next,we measured p65-NF-κB binding to the HOTAIR promoter in A2780p cells treated with CDDP in the presence and absence of Bay-11.Enrichment (5.2-fold)of p65in the canonical binding site 4was observed compared with control primer (Figure 5c)and abolished in the presence of Bay-11.To further examine the transcriptional activation of HOTAIR by p65-NF-κB,transient transfection assays wereperformed in 293cells using wild-type or mutant (906-CTCATTCTCA-915)sequences of HOTAIR promoter along with renilla vector.As shown in Supplementary Figure S4B,treat-ment with TNF-αincreased (P o 0.05)luciferase activity (1.6-fold)by the p65-NF-κB wild-type vector,no change in activity of the mutant promoter was observed,and TNF-α-induced luciferase activity was blocked (P o 0.05)by NF-κB inhibitor Bay-11.Regulation of HOTAIR expression by NF-κB during DDRTo examine the effect of additional DNA-damaging agents on NF-κB activation,we used a previously reported luciferasereporterFigure 2.DNA damage induces HOTAIR expression.(a )A2780p cells transfected with HOTAIR or empty vector or (b )A2780_CR5cells transfected with either dsiGFP or dsiHOT and were treated with CDDP (24h post transfection).Western blots show total Chk1and p-Chk1levels at the indicated times post-CDDP treatment at the same exposure for both cell types.Western is representative of three biological replicates.(c )A2780p cells transfected with HOTAIR or vector control or (d )A2780_CR5cells transfected with either dsiGFP or dsiHOT were untreated or treated with CDDP (10μM ).Cells were fixed and stained for γ-H2AX foci (red)at the indicated time points.Cells were counted at ×60magni fication (average of 100cells counted),and the total number foci were determined.All experiments were repeated three times and error bars represent s.d.(e )HOTAIR or vector expressing A2780p or A2780_CR5cells transfected with either dsiGFP or dsiHOT were treated with indicated concentration of CDDP for 3h,and 24h post treatment caspase 3/7cleavage assay was performed.Bar graph represents caspase 3/7cleavage normalized to untreated control.Western blot is representative of two biological replicates.Asterisks indicate P o 0.05(*)or P o 0.01(**).NF-κB-HOTAIR axis links DDR,chemoresistance and cellular senescenceAR Öze şet al4Oncogene (2016)1–12©2016Macmillan Publishers Limitedconstruct containing 861base pairs of the E-selectin promoter containing 3canonical NF-κB-p65-binding sites as a positive control.23TNF-αtreatment increased luciferase activity compared with empty vector (Supplementary Figure S4C),con firming luciferase activation by known NF-κB inducers.In cells treated with MMC,H 2O 2or CDDP,we observed increased luciferase activity,suggesting that various types of DNA damage can activate NF-κB (Supplementary Figure S4D).To determine NF-κB-speci fic transcription luciferase activity was determined in p65-wt or -mutant vector-transfectedA2780p cells (described in Supplementary Figure S4D)treated with MMC,H 2O 2or CDDP.As shown in Figure 5d,p65-wt HOTAIR promoter activity was increased by these genotoxic agents (6.9-,8.2-and 5.0-fold by MMC,CDDP or H 2O 2relative to empty vector,respectively)and was inhibited (P o 0.05)by pretreat-ment with Bay-11(Figure 5e).No change in luciferase activity was observed with the p65-mutant construct relative to wild-type vector (Figures 5d and e).In all,our results suggest that HOTAIR is transcriptionally regulated by NF-κB as a response to DNAdamage.Figure 3.HOTAIR overexpression induces DDR and NF-κB target gene expression.(a )Western blot showing total I κ-B αand p65-NF-κB levels in cytoplasmic and nuclear fractions in A2780and A2780-CR5cells.(b )Western blot showing total I κ-B αlevels in cells expressing vector control or HOTAIR post CDDP treatment (10μM )at indicated times.(c )A2780p cells transfected with HOTAIR or vector control were untreated or treated with CDDP (10μM )for the indicated times.Cells were fixed and stained for NF-κB p65(red).The total number of foci was determined based on the average of 100cells counted at ×60magni fication.Bar graph represents fold-change of p65translocation relative to untreated.All experiments were repeated three times and error bars represent s.d.(d )Western blot showing nuclear I κ-B αand NF-κB levels in A2780p cells expressing vector control or HOTAIR either post CDDP (10μM ),Bay-11(3μM )or combination treatment.(e )A2780_CR5cells transfected with either dsiGFP or dsiHOTAIR were untreated or treated with CDDP (10μM )for the indicated times.Cells were fixed and stained for NF-κB p65(red).The total number of foci was determined based on the average of 100cells counted at ×60magni fication.(f )Western blot showing nuclear I κ-B αand NF-κB levels in A2780_CR5transfected with either dsiGFP or dsiHOTAIR siRNA post CDDP (10μM ),BAY11(3μM )or combination treatment.NF-κB-HOTAIR axis links DDR,chemoresistance and cellular senescence AR Öze şet al5©2016Macmillan Publishers LimitedOncogene (2016)1–12Overexpression of HOTAIR induces IL-6secretion,DDR activation and cellular senescenceCytokines in the microenvironment contribute to DNA damage resistance 24and secretion of cytokines during persistent DNA damage has been reported.25IL-6secretion in particular was shown to contribute to a 'chemoresistant niche'.26Having observed signi ficant expression of interleukin receptors in A2780_CR5(Figure 3b),indicative of pathway activation,we performed an NF-κB target cytokine screen.HOTAIR overexpres-sion was associated with increased (42-fold)secretion of CCL5,IL-5,IL-6,CXCL-11and IL-23compared with vector control (Supplementary Figure S5A).Moreover,treatment of A2780p cells with 'HOTAIR-conditioned'media increased (P o 0.05)CDDP IC 50by 42-fold,which was reversed using an IL-6neutralizing antibody (Figure 6a).The addition of recombinant IL-6to unconditioned media resulted in an approximate twofold increase (P o 0.05)in the IC 50for CDDP (Figure 6b),and dsiRNA knockdown of HOTAIR in A2780_CR5cells reduced (P o 0.05)IL-6secretion (Figure 6c)and IC 50for CDDP (Figure 6d).Recombinant IL-5had no effect on CDDP IC 50(Supplementary Figure S5B),further indicating a role for HOTAIR-mediated IL-6induction in platinum-DNA damage response.Based on a recent report that unresolved DNA strand breaks caused by DNA damage followed by NF-κB activation and IL-6secretion can induce cellular senescence,25we examined whether treatment with low (1μM CDDP or 1/10th of IC 50)or high (20μM CDDP or 2×the IC 50)doses of CDDP induces senescence in A2780p cells overexpressing HOTAIR or not.Activation of p53and p21by HOTAIR was observed only during high CDDP treatment,indicated by p53phosphorylation and p21expression (Figure 6e).In addition,IL-6secretion,an established marker for cellsenescence,25,27was increased (P o 0.05)by CDDP in A2780p overexpressing HOTAIR compared with vector-transfected cells (Supplementary Figure S5C).Based on the observation that addition of recombinant IL-6increased (P o 0.05)proliferation (Supplementary Figure S5D),we reasoned that IL-6secretion by senescent cells induced proliferation of neighboring cells.We then investigated whether an increase in p-Chk1and p-p53was dependent on ATR signaling (ataxia telangiectasia mutated (ATM)and Rad3-related (ATR)signaling pathway).28In HOTAIR overexpressing vs vector control cells,decreased p-p53levels (Figure 7a,left)were observed and the level of p-Chk1was essentially unchanged (Figure 7a,right),suggesting ATR-dependent activation of p53by HOTAIR (further supported by no change in proliferation in the presence of ATRi;Supplementary Figure S5E).Next,we measured p-Chk1and p-p53levels in A2780_CR5cells upon dsiRNA knockdown HOTAIR and observed decreased p-Chk1and p-p53levels overall,and this effect was abolished in the presence of ATR inhibitor (Supplementary Figure S5F).To examine the lack of p65-NF-κB on cell proliferation and CDDP resistance,we stably knocked down p65-NF-κB and validated reduced luciferase activity after stimulation with TNF-α(Supplementary Figure S6A and B).Interestingly,ectopic expres-sion of HOTAIR in NF-κB knockdown cells rescued (P o 0.05)proliferation and increased (P o 0.05)clonogenic survival (Supplementary Figure S6C and D),which may be attributed to the previously described role of HOTAIR in inhibiting p21as a potential mechanism of chemotherapy resistance and evasion of apoptosis.29,30To examine cellular senescence,we measured β-galactosidase activity (SA-β-Gal).31In HOTAIR overexpressing A2780p cells,SA-β-Gal-positive cell numbers were increased (P o 0.05)by high vslowFigure 4.Transcriptional upregulation of HOTAIR by NF-κB during DNA damage.(a )HOTAIR expression in A2780p treated with CDDP alone or in combination with NF-κB inhibitor Bay-11(HOTAIR levels measured by qRT-PCR 24h post treatment).(b )HOTAIR expression in A2780p cells transfected with IKK-αand IKK-βand western blot showing co-expression of IKK αand IKK βin A2780p cells 24h post transfection.(c )Average expression (log 10fold-change)of NF-κB target genes STAT1,IL-6R,IL-6,IL-4R,IL8,IL1R1,BCL2L1,MMP-9in A2780p cells (red bars)HOTAIR expression (fold-change)was measured by normalizing to vector control.For A2780_CR5cells transfected with dsiHOTAIR (yellow bars),fold-change was measured by normalizing to dsiGFP-transfected cells.(d )Average expression of NF-κB target genes measured in mouse xenografts overexpressing HOTAIR (treated with CDDP compared with untreated).Fold-change was measured by normalizing to xenografts with vector control.Bars represent average measurements ±s.d.(n =4per group).Asterisks indicate P o 0.05(*)or P o 0.01(**).NF-κB-HOTAIR axis links DDR,chemoresistance and cellular senescenceAR Öze şet al6Oncogene (2016)1–12©2016Macmillan Publishers Limitedlevels of CDDP (Supplementary Figure S7A),and NF-κB inhibitor Bay-11reduced (P o 0.05)the number of senescent cells (Figure 7b),which is likely a cytostatic effect of the drug (and not cytotoxicity),based on the observation that ectopic expression of HOTAIR increased cell proliferation in cells treated with the NF-κB inhibitor (Supplementary Figure S5C).DsiRNA knockdown of HOTAIR in A2780_CR5cells reduced the number of senescent cells (Figure 7c).Next,we performed flow cytometry in both HOTAIR expressing A2780p and dsiHOTAIR knockdown A2780_CR5cells 24and 48h post CDDP treatment.We observe a decrease in S1and an increase in G2phase 48h post CDDP treatment in HOTAIR expressing cells (Figure 7d)and a reversal of this effect in A2780_CR5cells (Figure 7e),suggesting that a subpopulation of cells undergo HOTAIR-dependent cell senescence.Collectively,these results indicate that HOTAIR-induced NF-κB activation and IL-6secretion contributes to a senescent phenotype,and IL-6secretion in fluences the surrounding cell population in a paracrine manner,contributing to chemoresistance (Supplementary Figure S7C).DISCUSSIONHOTAIR is an lncRNA frequently overexpressed in solid tumors 32and associated with cancer cell growth and migration.Although HOTAIR overexpression in OC correlates with disease metastasisand poor patient prognosis,12,33,34the underlying mechanism of HOTAIR upregulation and the role of this important lncRNA in drug resistance in OC and other cancers are not fully understood.In this study,we demonstrate a key role for HOTAIR in DNA damage:HOTAIR modulates expression of genes activated during DDR,including Chk1and the levels of γ-H2AX,to inhibit cell-cycle progression.Our findings include that HOTAIR activation of the NF-κB pathway following CDDP-induced DNA damage contributes to cellular senescence.Furthermore,we demonstrate that DDR activation of NF-κB induces HOTAIR and a positive-feedback loop,resulting in sustained NF-κB activation and persistent DNA damage signaling.As platinum-based regimens continue to be the mainstay of treatment for many solid cancers including OC,35HOTAIR may represent a target for therapeutic intervention of drug resistance.Previous mapping studies revealed HOTAIR binding to chro-mosomal regions near the I κ-B αgene,supporting the potential regulation of NF-κB activity by HOTAIR.NF-κB is a master regulator of several cellular responses,including DDR,stress,senescence and in flammation,although its role in cancer is paradoxical,including both oncogenic and tumor suppressive activities.36In ovarian and other cancers,NF-κB activation is associated with chemoresistance 37,38and activation during DNA damage induced by genotoxic agents has been demonstrated but not well de fined.4,5We show that HOTAIR expression modulated NF-κBFigure 5.Direct transcriptional regulation of HOTAIR by NF-κB.(a )Map of HOTAIR promoter region showing canonical p65binding site (908bases upstream of TSS)and the mutant generated in PGL3vector.(b )ChIP in A2780_CR5cells of NF-κB p65targeting five regions upstream of HOTAIR TSS (1:10kb,2:8kb,3:5kb,4:908bp (consensus p65site)and 5:203bp).Asterisks indicate P o 0.05(*)or P o 0.01(**).(c )ChIP in A2780_CR5cells of NF-κB p65post CDDP (10μM )or in combination with BAY11(3μM )targeting regions 1and 4upstream of HOTAIR TSS (1:10kb,4:908bp (consensus p65site)with primer corresponding to IL8promoter as a (+)control.(d )Luciferase activity of HOTAIR promoter (illustrated in a )in A2780p cells treated with MMC (10μM ),cisplatin (CDDP ,10μM )or hydrogen peroxide (H 2O 2,0.5m M ).(e )Luciferase activity of NF-κB p65wild-type or mutant HOTAIR promoter (Figure 4c)in A2780p cells pre-treated Bay-11(5μM for 1h)then treated with MMC (10μM ),CDDP (10μM )or H 2O 2(0.5m M ).Asterisks indicate P o 0.05(*)or P o 0.01(**).NF-κB-HOTAIR axis links DDR,chemoresistance and cellular senescence AR Öze şet al7©2016Macmillan Publishers LimitedOncogene (2016)1–12。